Since a long time ago, cellular metabolisms such as microbial fermentation have been used to process food. However, as the understanding of intracellular mechanisms has been enhanced, it became possible to regulate cellular metabolic pathways using DNA recombination technology and a variety of recently developed new synthetic devices in order to satisfy engineering demands. Synthetic systems can be used without difficulty to introduce phenotypic perturbation, because artificial operations are operated independently of intracellular regulatory systems. Thus, such synthetic systems have the potential to widen the range of accessible phenotypes. As a good example of this case, synthetic riboswitches have been artificially designed and introduced into microorganisms in order to reprogram microbial metabolic pathways according to engineering demands.
Riboswitches are constructs that regulate genes in a small ligand-dependent manner, and have been found in a variety of RNAs.
Natural riboswitches modify their structures in response to the concentration of an intracellular metabolite, in which the metabolite (ligand) binds to sites other then the active site of the riboswitch (allosteric binding) to modify the three-dimensional structure of the RNA, thereby regulating the expression of the gene. As a result, the termination of transcription of the gene occurs faster than normal, or the initiation of translation into protein is prevented, or the degradation rate of mRNA is affected by the modification.
In view of the fact that riboswitches are natural metabolite sensors, a synthetic genetic circuit comprising a synthetic riboswitch can be developed and applied in terms of evolutionary engineering in order to achieve the high-efficiency screening of microorganisms that produce useful metabolites. In order to efficiently screen mutants, which overproduce useful metabolites, from mutants having randomly perturbed phenotypes, the mutants should be capable of showing the change in color as a result of the production of the metabolites such that the phenotype of the mutants can be visually observed, or the mutants should be capable of showing the change in the growth rate according to the production of the metabolites. However, only several kinds of metabolites show natural color development or fluorescence, and most metabolites do not show this phenotype. Due to this limitation, there is a problem in that inefficient screening that requires complex experimental procedures is performed. In order to overcome this limitation when screening invisible phenotypes, several attempts have been made to use synthetic metabolite sensors. Particularly, a synthetic riboswitch coupled with a fluorescence protein reporter was developed, and as demonstrated by fluorescence-activated cell sorting (FACS), this synthetic riboswitch efficiently induces the evolution of the enzyme caffeine demethylase (Michener J K et al., Metab Eng 14:306-316, 2012).
However, it was found that screening of mutants showing a fast growth rate shows the highest efficiency (Dietrich J A et al., Annu Rev Biochem 79:563-590, 2010). In view of this fact, the present inventors have designed a synthetic genetic circuit showing growth rate dependence. Specifically, the synthetic genetic circuit was designed such that it would be possible to select a mutant that overproduces a target metabolite as a result of the change in the growth rate of cells with a change in the concentration of the metabolite in the cells.
In addition, the present inventors have made extensive efforts to enable this screening principle to be applied to various strains that produce various metabolites, and as a result, have designed a riboswitch-based synthetic suicide genetic circuit based on two general and common cellular mechanisms.
One mechanism is an mRNA degradation mechanism in which a self-cleaving ribozyme is involved. This mechanism has already been applied to many genetic circuits, and it is known that this mechanism can be widely applied not only to eukaryotic cell systems, but also to prokaryotic cell systems.
The other mechanism is a generally known mechanism in which the conversion of fluorocytosine to fluorouracil by cytosine deaminase induces cell death (apoptosis). As is known in the art, this mechanism is because fluorouracil, an analog of uracil, exhibits toxicity in eukaryotic cells and prokaryotic cells, resulting in dysfunction of RNA.
In view of these facts, the present inventors have found that for example, the use of a suicide genetic circuit comprising the ribozyme glmS (which responds to the intracellular concentration of the yeast metabolite glucosamine-6-phosphate (GlcN6P)) inserted into the cytosine deaminase-encoding gene FCY1 makes it possible to efficiently screen only yeast mutants that overproduce GlcN6P, thereby completing the present invention.